Prior art methods for high-sensitivity detection of chemical and biological materials have typically relied on colorimetric or electrometric analyses of enzymatic or immunological reactions of the test analytes. Enzyme immunoassay (EIA) techniques have been incorporated into a large number of biosensor devices employing colorimetric or fluorimetric analyses, as exemplified in the following U.S. and foreign patents: U.S. Pat. No. 4,343,782; EP 125,554; EP 128,318; EP 231,010; EP 288,256; EP 290,269; SU 1,189,224; JP 57,208,457. In a typical embodiment, an enzyme-conjugated antigen admixed with immobilized antibody reacts competitively with free test antigen to release the enzyme into solution containing its substrate, and subsequent enzyme reaction products are detected by changes in color or fluorescence. A drawback of this method is background color which limits sensitivity.
U.S. Pat. No. 4,343,782 to Shapiro, issued Aug. 10, 1982, describes a cytological assay procedure for non-excitable cells in which their membrane potential measured by an optical property of a permeant dye is used to determine characteristics of individual cells. Japanese Patent No. 57,208,457 to Olympus Optical Co., Ltd., published Dec. 21, 1982, describes an automated apparatus for staining of cells or tissues, including an indicator installed for the determination of dye concentration.
European Patent No. 125,554 to Charlton, published Nov. 21, 1984, describes an ion test means having a hydrophilic carrier matrix, which consists of a hydrophilic carrier matrix loaded with hydrophobic globules containing an ionophore, and a reporter substance and titanium dioxide, in a test strip attached to a support.
European Patent No. 128,318 to Charlton et al., published Dec. 19, 1984, describes the use of substituted indonaphthols as reporter substances in detecting ions in ionophore-containing test strips, for the detection of ions in aqueous solutions. U.S.S.R. Patent No. 1,189,224 to Lemeshko and Brovkovich, published Jul. 26, 1983, describes a method of determining phospholipids in biological membranes, by treating the preparations with chloroform-methanol and a molybdenum color reagent.
European Patent No. 231,010, to Halsey et al., published Aug. 5, 1987, describes a method of solid phase enzyme immunoassay and nucleic acid hybridization assay, in which a chromogenic material upon changing color in the presence of a solid phase enzyme, binds to the solid phase. Color change is analyzed on a dip stick, for use in allergy testing. European Patent No. 288,256, to Toner, published Oct. 26, 1988, describes europium chelates with polypyridine and phenanthroline derivatives for fluorescent labels for immunoassays.
Electrochemical coupling of EIA permits direct electronic readout, but may suffer from similar sensitivity limitations. Examples of prior art methods are disclosed in the following U.S. and foreign patents: U.S. Pat. No. 4,214,968; JP 58,103,659; JP 60, 44,865; JP 62,64,941; JP 63,171,355; JP 63,263,468; EP 302,661. U.S. Pat. No. 4,214,968 to Battaglia et al., issued July 29, 1980, describes a dry operative ion selective electrode comprising a dried salt solution in a hydrophilic polymeric binder in the internal reference, in contact with a hydrophobic ion-selective membrane containing an ion carrier dissolved therein.
Japanese Patent No. 60,44,865 to Fuji Photo Film Co., Ltd., published Mar. 11, 1985, describes a multilayered analytical element for ammonia determination in body fluids comprising a water-resistant support coated with an ammonia indicator layer, a barrier layer which is permeable to ammonia but not to liquid substances, a reagent layer which transforms ammonia-forming substances to ammonia, and a porous spreading layer. Japanese Patent No. 58,103,659 to Toshiba Corp., published Jun. 20, 1983, describes membranes for ion-selective electrodes, including a tetrahydrofuran solution of valinomycin dispersed in polyvinyl chloride in the presence of dioctyl phthalate and potassium tetraphenylboron, dried into a membrane useful for the determination of ions in blood. Japanese Patent No. 63,171,355 to Tsukada et al., published Jul. 15, 1988, describes a semiconductor chemical sensor for blood analysis, in which the surface of a field effect transistor gate on a base is coated with a hydrophilic polymer membrane and the membrane is treated with electrically accelerated particles to form hydrophilic groups.
Japanese Patent No. 62,64,941 to Takei et al., published Mar. 24, 1987, describes enzyme-immobilized membranes for a sensor, in which high molecular weight substances are dissolved, the mixture spread and air dried, and soaked in a solvent in which the high molecular weight component is insoluble, for enzyme immobilization. Japanese Patent No. 63,263,460 to Mitsumata et al., published Oct. 31, 1988, describes an immunoassay method using an enzyme immunosensor electrode, in which an electrode containing immobilized antibody is inserted into an electrolyte solution, enzyme-labelled antigen and a test antigen are added for competitive reaction with antibody, and instantaneous current or potential changes measured for detection of the antigen. European Patent No. 302,661 to Vadgama et al., published Feb. 8, 1989, describes a supported liquid membrane enzyme electrode sensor for analyte determination.
Biosensors incorporating electrochemical detection with adsorbed, deposited, or embedded receptor agents on an electrode are exemplified in the following U.S. and foreign patents: U.S. Pat. No. 4,634,599; U.S. Pat. No. 4,661,442; JP 63,153,462; JP 63,206,652; JP 01,59,058; EP 304,447; WO 87,03,095; WO 88,08,972; WO 88,09,499; and WO 88,09,808. In these systems, analyte detection is typically followed by a change in electrical potential or current at an electrode surface. Sensitivity and selectivity for specific analytes depend on the nature of the materials used on the electrode, and have limited the applicability of these devices.
Japanese Patent No. 63,153,462 to Takei et al., published Jun. 25, 1988, describes a sensitive field effect transistor biosensor containing an aluminum gate-immobilized biological substance. European Patent No. 304,947 to Kagayama, published Mar. 1, 1989, describes a biosensor containing immobilized physiologically active substance and transducer, comprising a pair of opposing flat plates between which an antibody, with antigen or enzymes immobilized onto one surface and a transducing chemical substance on the other surface.
PCT International Application No. WO 88,08,972 to Cheung et al., published Nov. 17, 1988, describes a biosensor comprising a reversibly selective binding protein immobilized upon the insulated gate region of a field effect transistor on the sensor. PCT International Application No. WO 88,09,808 to Taylor and Marenchic, published Dec. 15, 1988, describes receptor-based biosensors and a method of immobilizing and stabilizing an active biological receptor in a polymeric film onto an electrode. PCT International Application No. WO 88,09,499 to Newman, published Dec. 1, 1988, describes an optimized capacitive sensor for chemical analysis, which relies on biospecific binding between a biochemical binding system and the analyte of interest to change the dielectric properties of a capacitive affinity sensor.
Japanese Patent No. 01,59,058 to Kuriyama, published Mar. 6, 1989, describes an enzyme immunosensor and its use in enzyme immunoassay, consisting of a semiconductor ion sensor, a spacer, and plate containing immobilized antigen or antibody. French Patent 2,614,422 to Liston et al., published Oct. 28, 1988, describes an enzyme electrode and module for determination of analytes in physiological fluids, comprising a membrane chamber and composite layered membrane, an electrode element in contact with the membrane, a flow sampler connected with the membrane chamber, a wash cell and a sample container, and means for intermittent transfer of sample and sample and other solutions. German Patent No. 3,226,045 to Seshimoto et al., published Jan. 20, 1983, describes film-like ion selective electrodes comprised of a conductive layer adjacent to an ion-selective layer, and their use for body fluid analysis.
PCT International Application No. WO 87,03,095 to Newman, published May 21, 1987, describes a capacitive affinity sensor and method for chemical analysis and measurement, comprising an open capacitor which produces a higher electric field in one volumetric region and a lower field in a second region, a biospecific binding agent for the analyte localized on the surface between the conductors in the first region, and a means associated with the capacitor which responds to the average dielectric constant in the first chamber. U.S. Pat. No. 4,634,599, to Uzgiris, issued Jan. 6, 1987, describes a method for making ordered monolayers of macromolecules on supported lipid polylayers, for two-dimensional crystallization of macromolecules for imaging in electron microscopy. U.S. Pat. No. 4,661,442, to Lukens, issued Apr. 28, 1987, describes a process for preparing lipid-protein membranes for chemical detection, comprising an aqueous medium containing emulsified lipid and protein, applying the medium to an orifice, and evaporating the water to provide a stable membrane.
Membrane electrode biosensors incorporating oxidative probes, such as those used to detect glucose in blood, are exemplified in the following patents: FR 2,614,422; EP 248,680; JP 62,85,853; JP 01,59,055; JP 01,60,382. In these systems, a Clark-type oxygen sensing electrode is typically coupled to an oxidative enzyme such as glucose oxidase, in order to permit analysis of the desired biological parameter. Among other factors, synthetic membranes used in said systems vary in composition among the biosensor devices.
Japanese Patent No. 01,60,382 to Asakura, published Mar. 7, 1989, describes the manufacture of a fibroin-enzyme membrane and its use in an enzyme sensor. Japanese Patent No. 01,59,055 to Suetsugu et al., Mar. 6, 1989, describes water-absorbing layers in biosensors, for electrochemical detection of substances based on a reaction with a redox enzyme and an electron acceptor. Japanese Patent No. 62,85,853 to Miyai and Asano, published Apr. 20, 1987, describes a membrane for an enzyme electrode, comprised of an enzyme such as glucose oxidase immobilized on a fine powder carrier and supported with a membrane having a fractionating molecular weight capable of passing the protein molecule of the immobilized enzyme.
European Patent No. 248,680 to Reinhart et al., published Dec. 9, 1987, describes a noninvasive electrochemical apparatus and method for determining blood glucose, comprising a membrane means for containing glucose oxidase, means for providing oxygen to the membrane, and means for measuring the hydrogen peroxide product in the membrane.
Sensors using gated membrane electrodes, in which a reactive material for the analyte of interest is included in a membrane whose permeability changes upon reaction with the analyte, are exemplified in the following U.S. and foreign patents: U.S. Pat. No. 4,637,861; U.S. Pat. No. 4,776,944; and EP 261,887. Their sensitivity and selectivity are determined by the gated membrane composition, and can limit the types of agents which one can analyze by these methods. Activity of the gate material in prior art membranes can be strongly influenced by subtle physical changes in the membrane. For example, Krull, U. J., M. Thompson, E. T. Vandenberg, and H. E. Wong (1985) "Langmuir Blodgett Film Characteristics and Phospholipid Membrane Ion Conduction, " Analytica Chimica Acta 174: 83-94; 95-102, describe packing density effects and capacitance measurements on electrodes with phospholipid membranes. Other influences include regulatory substances. For example Montal, M. P. Labarca, D. R. Fredkin, B. A. Suarez-Isla, and J. Lindstrom (1984) "Acetylcholine Receptor from Torpedo californica Reconstituted in Planar Lipid Bilayer Membranes," Biophysical J. 45: 165-174, describe effects of bungarotoxin and other ion channel modulating toxins in phospholipid membranes. See also Krueger, B. K., J. F. Worley, and R. J. French (1986) "Block of Sodium Channels in Planar Lipid Membranes by Guanidinium Toxins and Calcium: Are the Mechanisms of Voltage Dependence the Same?" Annals of the New York Academy of Sciences 479: 257-268).
Caras and Janata, "Enzymatically Sensitive Field Effect Transistors," Methods in Enzymology, Volume 137: pages 247-255, 1988, describes the design, fabrication, principles of operation, applications and theoretical limitations of enzymatically coupled field effect transistors as solid state biosensors.